Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations

ABSTRACT

Enriched neural stem and progenitor cell populations, and methods for identifying, isolating and enriching for neural stem cells using reagent that bind to cell surface markers, are provided.

CLAIM OF PRIORITY

[0001] This application claims priority under 35 U.S.C. § 119(e), toU.S. provisional patent application Serial No. 60/119,725, filed Feb.12, 1999.

TECHNICAL FIELD

[0002] This invention relates generally to enriched neural stem cell andprogenitor cell populations, and methods for identifying, isolating andenriching for neural stem and progenitor cells, particularly centralnervous system neural stem cells and progenitor cells, and mostparticularly to enriched populations of neurosphere initiating cells(NS-IC).

BACKGROUND OF THE INVENTION

[0003] Stem cell populations constitute only a small percentage of thetotal number of cells, but are of immense interest because of theirability to repopulate the body. The longevity of stem cells and thedissemination of stem cell progeny are desirable characteristics. Thereis significant commercial interest in these methods because stem cellshave a number of clinical uses. There is also medical interest in theuse of stem cells as a vehicle for gene therapy.

[0004] Proteins and other cell surface markers found on stem cell andprogenitor cell populations are useful in preparing reagents for theseparation and isolation of these populations. Cell surface markers arealso useful in the further characterization of these important cells.

[0005] Yin et al., U.S. Pat. No. 5,843,633, incorporated herein byreference, describes a monoclonal antibody called AC133, which binds toa surface marker glycoprotein on hematopoietic stem and progenitorcells. The AC133 antigen is a 5-transmembrane cell surface antigen witha molecular weight of 117 kDa. Expression of this antigen is highlytissue specific, and has been detected on a subset of hematopoieticprogenitor cells derived from human bone marrow, fetal bone marrow andliver, cord blood, and adult peripheral blood. The subset of cellsrecognized by the AC133 antibody is CD34^(bright), and containssubstantially all of the CFU-GM activity present in the CD34⁺population, making AC133 useful as a reagent for isolating andcharacterizing human hematopoietic progenitor and stem cells.

[0006] However, surface markers specific to non-hematopoietic stem cellsand progenitor cells, and particularly central nervous system neuralstem cells and progenitor cells have not been identified. Further, theAC133 antibody has not been used in methods for identifying, isolating,or enriching for non-hematopoietic stem cells or progenitor cells,particularly central nervous system (CNS) neural stem cells andprogenitor cells. There remains a need for tools, such as monoclonalantibodies that are useful in isolating and characterizing humannon-hematopoietic progenitor and stem cells, and particularly centralnervous system (CNS) neural stem cells and progenitor cells.

SUMMARY OF THE INVENTION

[0007] This invention provides methods for identifying, isolating, andenriching for human non-hematopoietic progenitor and stem cells, andparticularly central nervous system (CNS) neural stem cells which caninitiate neurospheres (NS-IC) and progenitor cells. The invention alsoprovides for enriched populations containing CNS neural stem cells thatcan initiate neurospheres, and progenitor cells. A “neurosphereinitiating cell (NS-IC)” is a cell that can initiate long-termneurosphere culture. A “neurosphere”, in turn, is an aggregate orcluster of cells which includes neural stem cells and primitiveprogenitors. The identification, culture, growth, and use ofneurospheres is disclosed in Weiss et al., U.S. Pat. No. 5,750,376 andWeiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. While the term “NS-IC” is defined by the ability or capacityof that cell to form a neurosphere, these cells may be appropriatelygrown in adherent culture (see, for example, Johe, U.S. Pat. No.5,753,506, incorporated herein by reference), and it should be notedthat the methods and populations described herein are not to be limitedto suspension cultures of NS-IC. An NS-IC is nestin⁺ and has thecapability to differentiate, under appropriate differentiatingconditions, to neurons, astrocytes, and oligodendrocytes.

[0008] According to one embodiment of this invention, enrichedpopulations of non-hematopoietic stem cells and progenitor cells,preferably CNS neural stem cells including NS-ICs, and progenitor cells,and method of identifying, isolating, or enriching for such cells, isachieved by contacting a population of cells containing at least onestem cell or NS-IC, or progenitor cell with a reagent that binds tosurface marker glycoprotein antigen (“AC133 antigen”) recognized by theAC133 antibody. In a preferred embodiment the reagent is the AC133antibody (the AC133 antibody is alternately referred to herein as“5F3”). Use of traditional techniques for cell sorting, such as byimmunoselection (e.g., FACS), then permits identification, isolation,and/or enrichment for cells in which contact between the reagent and theAC133 antigen has been detected.

[0009] In another embodiment, this invention provides a novel antibody,herein called 5E12, that may be used to provide enriched populations ofnon-hematopoietic stem cells and progenitor cells, preferably CNS neuralstem cells that can initiate neurospheres and progenitor cells, and maybe used in methods of identifying, isolating, or enriching for suchcells, by contacting a population of cells containing at least one stemcell NS-IC, or progenitor cell with the 5E12 antibody, which binds to asurface marker glycoprotein antigen other than the AC133 antigen.

[0010] In a preferred embodiment, the cells of this invention,preferably the CNS neural stem cells, are additionally characterized aslacking cell surface markers for CD45 and CD34.

[0011] In a further embodiment, this invention provides a novelantibody, herein called 8G1, believed to recognize CD24, which permitssubselection between populations of CNS neural stem cells (characterizedas 8G1^(−/lo)) and populations of CNS progenitor cells (characterized as8G1⁺).

BRIEF DESCRIPTION OF THE FIGURE

[0012]FIG. 1 is a diagram illustrating the proliferation anddifferentiation of a NS-IC.

[0013]FIG. 2 is a series of photographs showing that neurospherecultures can be initiated from single-cell sorted 5F3⁺ cells.

[0014]FIG. 3 is a dot plot of fluorescence activated cell sorting (FACS)data showing the isolation of human CNS neural stem cells using cellsurface markers using the monoclonal antibody 5E12. The x axisrepresents cell staining for antibodies to CD34 and CD45. The y axisrepresents cell staining with the 5E12 antibody.

[0015]FIG. 4 is a two panel dot plot of FACS sorting data showing theisolation of human neural stem cells by cell surface markers. Panel Ashows that 5F3⁺ cells co-express the antigen for the 5E12 antibody.Panel B shows that 5F3⁺ cells typically do not express the antigen forthe 8G1 antibody.

[0016]FIG. 5 is a chart showing the distribution of 5F3⁺ cells in fetalbrain as a function of gestational age.

[0017]FIG. 6 is a series of photographs showing results of thetransplantation of human neural cells into NOD SCID mouse.

[0018]FIG. 7 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the rostral migratory stream (RMS) whentransplanted into a rodent model.

[0019]FIG. 8 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the (RMS) into the olfactory bulb whentransplanted into a rodent model.

DETAILED DESCRIPTION OF THE INVENTION

[0020] A population of cells exists within the adult central nervoussystem (CNS) which exhibit stem cell properties, in their ability toself-renew and to produce the differentiated mature cell phenotypes ofthe adult CNS. These stem cells are found throughout the CNS, andparticularly in the subventricular regions, and dentate gyms of thehippocampus.

[0021] Growth factor-responsive stem cells can be isolated from manyregions of the neuraxis and at different stages of development, ofmurine, rodent and human CNS tissue. These cells vary in their responseto growth factors such as EGF, basic FGF (bFGF, FGF-2) and transforminggrowth factor alpha (TGF∝), and can be maintained and expanded inculture in an undifferentiated state for long periods of time. Bothadult and embryonic murine progenitor cells respond to EGF and grow asspheres of undifferentiated cells. These cells show the characteristicsof stem cells in that they are multipotent, and under serum containingconditions can differentiate into neurons, astrocytes andoligodendrocytes, as well as maintaining a subpopulation which remainsundifferentiated and continues to proliferate under EGF administration.Murine EGF-responsive progenitor cells express mRNA for the EGF receptorin vitro. Human CNS neural stem cell cultures have also been identified.The identification, culture, growth, and use of mammalian, includinghuman, neural stem cell cultures, either as suspension cultures or asadherent cultures, is disclosed in Weiss et al., U.S. Pat. No. 5,750,376and Weiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. Similarly, Johe, U.S. Pat. No. 5,753,506, incorporated hereinby reference, refers to adherent CNS neural stem cell cultures. Whencultured in suspension, CNS neural stem cell cultures typically formneurospheres.

[0022]FIG. 1 is shows the proliferation of a NS-IC as it develops into aneurosphere, and subsequent differentiation into neuronal and glialphenotypes, as well as generation of a progeny NS-IC. In the presence ofone or more proliferation-inducing growth factors, the NS-IC divides andgives rise to a sphere of undifferentiated cells composed of more stemcells and progenitor cells (a “neurosphere”). When the clonally derivedneurosphere is dissociated and plated as single cells, in the presenceof one or more proliferation-inducing growth factors, each NS-IC cangenerate a new neurosphere. The cells of a single neurosphere are clonalin nature because they are the progeny of a single neural stem cell. Inthe continued presence of a proliferation-inducing growth factor such asEGF or the like, precursor cells within the neurosphere continue todivide resulting in an increase in the size of the neurosphere and thenumber of undifferentiated neural cells. The neurosphere is notimmunoreactive for glial fibrillary acidic protein (GFAP; a marker forastrocytes), neurofilament (NF; a marker for neurons), neuron-specificenolase (NSE; a marker for neurons) or myelin basic protein (MBP; amarker for oligodendrocytes). However, cells within the neurosphere areimmunoreactive for nestin, an intermediate filament protein found inmany types of undifferentiated CNS cells (Lehndahl et al., 60 CELL585-595 (1990), incorporated herein by reference). Antibodies areavailable to identify nestin, including the rat antibody referred to asRat401. If the neurospheres are cultured in conditions that allowdifferentiation, the progenitor cells differentiate to neurons,astrocytes and oligodendrocytes. The mature phenotypes associated withthe differentiated cell types that may be derived from the neural stemcell progeny are predominantly negative for the nestin phenotype.

[0023] The terminology used for undifferentiated, multipotent,self-renewing, neural cells has evolved such that these cells are nowtermed “neural stem cells.” A neural stem cell is a clonogenicmultipotent stem cell which is able to divide and, under appropriateconditions, has self-renewal capability for NS-IC and can include in itsprogeny daughter cells which can terminally differentiate into neurons,astrocytes, and oligodendrocytes. Hence, the neural stem cell is“multipotent” because stem cell progeny have multiple differentiationpathways. A neural stem cell is capable of self maintenance, meaningthat with each cell division, one daughter cell will also be on averagea stem cell.

[0024] The non-stem cell progeny of a neural stem cell are typicallyreferred to as “progenitor” cells, which are capable of giving rise tovarious cell types within one or more lineages. The term “neuralprogenitor cell” refers to an undifferentiated cell derived from aneural stem cell, and is not itself a stem cell. Some progenitor cellscan produce progeny that are capable of differentiating into more thanone cell type. For example, an O-2A cell is a glial progenitor cell thatgives rise to oligodendrocytes and type II. astrocytes, and thus couldbe termed a “bipotential” progenitor cell. A distinguishing feature of aprogenitor cell is that, unlike a stem cell, it does not exhibit selfmaintenance, and typically is thought to be committed to a particularpath of differentiation and will, under appropriate conditions,eventually differentiate into glia or neurons.

[0025] The term “precursor cells” refers to the progeny of neural stemcells, and thus includes both progenitor cells and daughter neural stemcells.

[0026] Cell markers. This invention provides for the identification,isolation, enrichment, and culture of neural stem cells that are capableof forming neurospheres (NS-IC). NS-ICs are identified or selectedthrough the binding of antigens, found on the surfaces of NS-ICs, toreagents that specifically bind the cell surface antigen.

[0027] One of these antigens is an antigen that binds to the AC133monoclonal antibody. The AC133 antibody (herein referred to as the 5F3antibody) is exemplary of antibody embodiments of reagents thatrecognize a human cell marker termed prominin. Prominin is a polytopicmembrane protein expressed in various epithelial cells (Weigmann et al.,94(23) Proc Natl Acad Sci USA. 12425-30 (1997); Corbeil et al., 112 (Pt7) J Cell Sci. 1023-33 (1999); Corbeil et al., 91(7) Blood 2625-6(1998); Miriglia et al., 91(11) Blood 4390-1 (1998)). Various AC133antibodies are described in U.S. Pat. No. 5,843,333, incorporated hereinby reference. A deposit of the murine hybridoma cell line AC133 was madeat the American Type Tissue Collection, 12301 Parklawn Drive, RockvilleMd. 20852, on Apr. 24, 1997, and given the ATCC designation HB12346.These AC133 antibodies are capable of immunoselection for the subset ofhuman cells of interest in this invention. Preferred AC133 monoclonalantibodies can be obtained commercially from Miltenyi Biotec Inc.(Auburn Calif.), including AC133/1-PE antibody (Cat #808-01) andAC133/2-PE antibody (Cat #809-01). For MACS separation, a 50:50 mixtureof the monoclonal antibodies is preferred. The high tissue specificityof AC133 expression is particularly advantageous during enrichment forhighly purified NS-IC populations.

[0028] 5E12 is a novel monoclonal antibody generated againstenzymatically-dissociated human fetal brain cells. The 5E12 monoclonalantibody was generated substantially according to the contralateralimmunization method described in Yin, U.S. Pat. No. 5,843,633,incorporated herein by reference. The antigen to which 5E12 binds has aputative MW 125 kD, and is currently believed to be a distinct antigenfrom prominin.

[0029] CD45 is the T200/leucocyte common antigen. Antibodies to CD45 arecommercially available. In a preferred embodiment, the cells of thisinvention and cultures containing them, are additionally characterized(in addition to being prominin positive) as lacking cell surface markerssuch as CD45.

[0030] CD34 is also known as gp105-120. Monoclonal antibodies to CD34are commercially available, and CD34 monoclonal antibodies have beenused to quantitate and purify lymphohematopoietic stem/progenitor cellsfor research and for clinical bone marrow transplantation.

[0031] The monoclonal antibody 8G1 is believed to recognize CD24(antibodies to CD24 are commercially available), and specifically reactswith the 515 kilodalton α-chain of human LRP/A2MR which is expressed ina restricted spectrum of cell types. A strong immunohistochemicalreaction is seen in hepatocytes, tissue macrophages, subsets of neuronsand astrocytes in the central nervous system, fibroblasts, smooth musclecells, and monocyte-derived foam cells in atherosclerotic lesions in thearterial wall. The antibody can also be used for the characterization ofa subset of myelomonocytic subtypes of chronic and acute leukemia(CD91). Antibodies to CD91 are commercially available.

[0032] Isolation, enrichment, and selection of cells. The population ofcells from which NS-ICs are isolated can be a neural tissue, apopulation of cells is dissociated from neural tissue, or a populationof cells in cell culture, e.g., a cells in a neurosphere culture or anadherent neural stem cell culture.

[0033] The invention provides for the isolation and identification ofNS-ICs. Identification of a neurosphere initiating stem cell (NS-IC)involves contacting a population of neural cells (or which containsneural or neural derived cells) with a reagent that binds to AC133antigen and detecting the contact between the reagent that binds toAC133 antigen and the AC133 antigen on the surface of cells. Those cellsto which the reagent binds are identified as NS-ICs. The identity ofthose cells can be confirmed by assays to demonstrate that the cells arein fact NS-ICs, capable of neurosphere initiation, self renewal andmultipotentcy.

[0034] The methods of this invention can also be used to isolate AC133⁺cells from AC133⁻ cells using an AC133 antibody, by combining apopulation of neural cells which contains a fraction of NS-ICs with areagent that specifically binds to the AC133 antigen, and then selectingfor AC133⁺ cells, to produce a selected population enriched in AC133⁺NS-ICs as compared with the population of neural cells before selection.

[0035] Accordingly, the invention further provides for the enrichment ofNS-ICs from neural tissue or neural stem cell cultures (e.g.,neurosphere suspension cultures or neural stem cell adherent cultures).The invention is thus useful for the enrichment of NS-IC from neuraltissue in which stem cells and progenitor cells occur at low frequency,or may have been depleted, such as late embryo, juvenile, and adulttissue. One of skill in the art can combine a population of neural cellscontaining a fraction of NS-ICs with a reagent that specifically bindsto the AC133 antigen; and select for the AC133⁺ cells. In this way, theselected AC133⁺ cells are enriched in the fraction of NS-IC as comparedwith the population of neural cells.

[0036] The cell selection can be by any suitable means known in the art,including flow cytometry, such as by fluorescence activated cell sortingusing a fluorochrome conjugated AC133 antibody. The selection can alsobe by high gradient magnetic selection using AC133 antibody isconjugated to magnetic particles. Any other suitable method includingattachment to and disattachment from solid phase, is also contemplatedwithin the scope of the invention.

[0037] One of skill in the art can derive the population of cells byimmunoselection using an AC133 antibody. The population of cells shouldcontain at least 30% AC133⁺ NS-ICs, preferably at least 50-70% AC133⁺NS-ICs, and more preferably greater than 90% AC133⁺ NS-ICs. Mostpreferable would be a substantially pure population of AC133⁺ NS-ICs,comprising at least 95% AC133⁺ NS-ICs. The degree of enrichmentobtained, and actually used, depends on a number of factors, includingthe method of selection, the method of growth, and the cell dose of thecells that are placed in culture for the initiation of neurospheres.

[0038] The population of cells can be derived from late embryo,juvenile, or adult mammalian central nervous system (CNS) tissue, or itmay be derived from existing cultures of neural stem cells, as describedin Weiss, U.S. Pat. No. 5,750,376, or Johe, U.S. Pat. No. 5,753,506. Inthe most preferred embodiment, the NS-IC are human. In some embodiments,the AC133⁺ cells in the population can be complexed to endothelialcells.

[0039] The in vitro cell cultures described herein containing anenriched population of AC133⁺ NSICs are generally characterized in thatthe cultures stain positive for nestin and, in the presence ofdifferentiation-inducing conditions, produce progeny cells thatdifferentiate into neurons, astrocytes, and oligodendrocytes.

[0040] One of skill in the art can introduce an isolated AC133⁺ cell toa culture medium, proliferate the isolated AC133⁺ cell in culture;particularly as a neurosphere; culture the progeny of the isolatedAC133⁺ cell under conditions in which the isolated AC133⁺ celldifferentiates to neurons, astrocytes, and oligodendrocytes; then detectthe presence of neurons, astrocytes, and oligodendrocytes. The presenceof neurons, astrocytes, and oligodendrocytes characterizes the isolatedAC133⁺ cell as an NS-IC.

[0041] Typically AC133⁺ NS-IC is cultured in a medium that permits thegrowth and proliferation of neurospheres. The culture in which theisolated AC133⁺ cell proliferates can be a serum-free medium containingone or more predetermined growth factors effective for inducingmultipotent neural stem cell proliferation. The culture medium can besupplemented with a growth factor selected from leukocyte inhibitoryfactor (LIF), epidermal growth factor (EGF), basic fibroblast growthfactor (FGF-2; bFGF) or combinations thereof. The culture medium can befurther supplemented with neural survival factor (NSF) (Clonetics,Calif.). The conditions in which the AC133⁺ cell differentiates toneurons, astrocytes, and oligodendrocytes can be culturing the AC133⁺cell progeny on a laminin-coated surface in culture medium containingfetal bovine serum (FBS) without EGF, FGF-2 or LIF.

[0042] The invention also provides a method for identifying the presenceof a growth factor that affects the growth of NS-IC. One of skill in theart combines a composition suspected of containing at least one growthfactor that affects the growth of NS-IC with a composition comprisingNS-IC, then determines the growth of the NS-IC as a function of thepresence of the composition. Altered (increased, decreased, etc.) NS-ICgrowth indicates the presence in the composition of a growth factor thataffects the growth of NS-IC. One can then further identify the growthfactor.

[0043] Antibodies to AC133. Antibodies to AC133 may be obtained orprepared as discussed in U.S. Pat. No. 5,843,633, incorporated herein byreference. The AC133 antigen can be contacted with an antibody, such asvarious AC133 monoclonal antibodies, which have specificity for theAC133 antigen. An AC133 antibody is characterized by binding to theAC133 protein under Western blot conditions from reducing SDS-PAGE gels.The AC133 antigen has a molecular weight, based on commerciallyavailable standards, in the range of about 117 kDa. The AC133 antigen isexpressed on a subset of progenitor cells derived from human bonemarrow, fetal bone marrow and liver, cord blood, and adult peripheralblood.

[0044] Antibodies to AC133 antigen can be obtained by immunizing axenogeneic immunocompetent mammalian host (including murine, rodentia,lagomorpha, ovine, porcine, bovine, etc.) with human progenitor cells.The choice of a particular host is primarily one of convenience. Asuitable progenitor cell population for immunization can be obtained byisolating CD34⁺ cells from cytokine mobilized peripheral blood, bonemarrow, fetal liver, etc. A suitable progenitor cell population forimmunization can be obtained from CNS neural stem cells or other NS-IC.Immunizations are performed in accordance with conventional techniques,where the cells may be injected subcutaneously, intramuscularly,intraperitoneally, intravascularly, etc. Normally, from about 10⁶ to 10⁸cells will be used, which may be divided up into 1 or more injections,usually not more than about 8 injections, over a period of from aboutone to three weeks. The injections may be with or without adjuvant, e.g.complete or incomplete Freund's adjuvant, specol, alum, etc.

[0045] After completion of the immunization schedule, the antiserum maybe harvested in accordance with conventional ways to provide polygonalantisera specific for the surface membrane proteins of progenitor cells,including the AC133 antigen. Lymphocytes are harvested from theappropriate lymphoid tisue, e.g. spleen, draining lymph node, etc., andfused with an appropriate fusion partner, usually a myeloma line,producing a hybridoma secreting a specific monoclonal antibody.Screening clones of hybridomas for the antigenic specificity of interestis performed in accordance with conventional methods.

[0046] AC133 antibodies can be produced as a single chain, instead ofthe normal multimeric structure. Single chain antibodies are describedin Jost et al., 269 J. BIOL. CHEM. 26267-73 (1994), incorporated hereinby reference, and others. DNA sequences encoding the variable region ofthe heavy chain and the variable region of the light chain are ligatedto a spacer encoding at least about 4 amino acids of small neutral aminoacids, including glycine or serine. The protein encoded by this fusionallows assembly of a functional variable region that retains thespecificity and affinity of the original antibody.

[0047] AC133 antibodies can be produced by use of Ig cDNA forconstruction of chimeric immunoglobulin genes (Liu et al., 84 PROC.NATL. ACAD. SCL. 3439 (1987) and 139 J. IMMUNOL. 3521 (1987),incorporated herein by reference. mRNA is isolated from a hybridoma orother cell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al., “Sequences ofProteins of Immunological Interest” N.I.H. PUBLICATION No. 91-3242(1991). Human C region genes are readily available from known clones.The chimeric, humanized antibody is then expressed by conventionalmethods.

[0048] AC133 antibodies can be produced as antibody fragments, such asFv, F(ab′)₂ and Fab. Antibody fragments may be prepared by cleavage ofthe intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CHI domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0049] Immunostaining. Biological samples are assayed for the presenceof AC133⁺ cells by any convenient immunoassay method for the presence ofcells expressing the surface molecule bound by the subject antibodies.Assays may be performed on cell lysates, intact cells, frozen sections,etc. The antibodies available from Miltenyi Biotec Inc. (Auburn Calif.)are suitable for the direct immunofluorescent staining of cells.

[0050] Cell sorting. The use of cell surface antigens to NS-IC cellsprovides a means for the positive immunoselection of progenitor cellpopulations, as well as for the phenotypic analysis of progenitor cellpopulations using flow cytometry. Cells selected for expression of AC133antigen may be further purified by selection for other stem cell andprogenitor cell markers.

[0051] For the preparation of substantially pure progenitor and stemcells, a subset of progenitor cells is separated from other cells on thebasis of AC133 binding. Progenitor and stem cells may be furtherseparated by binding to other surface markers known in the art.

[0052] Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography and “panning”with antibody attached to a solid matrix, e.g. plate, or otherconvenient technique. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Dead cellsmay be eliminated by selection with dyes associated with dead cells(propidium iodide [P1], LDS). Any technique may be employed which is notunduly detrimental to the viability of the selected cells.

[0053] Conveniently, the antibodies are conjugated with labels to allowfor ease of separation of the particular cell type, e.g. magnetic beads;biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry. Multi-color analysis is of interest for the separation ofcells based on multiple surface antigens, e.g. AC133⁺ CD45⁻, AC133⁻CD34⁺, etc. Fluorochromes which find use in a multi-color analysisinclude phycobiliproteins, e.g. phycoerythrin and allophycocyanins;fluorescein and Texas red. A negative designation indicates that thelevel of staining is at or below the brightness of an isotype matchednegative control. A dim designation indicates that the level of stainingmay be near the level of a negative stain, but may also be brighter thanan isotype matched control.

[0054] In one embodiment, the AC133 antibody is directly or indirectlyconjugated to a magnetic reagent, such as a superparamagneticmicroparticle (microparticle). Direct conjugation to a magnetic particleis achieved by use of various chemical linking groups, as known in theart. Antibody can be coupled to the microparticles through side chainamino or sufhydryl groups and heterofunctional cross-linking reagents. Alarge number of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyidithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

[0055] Alternatively, AC133 antibody is indirectly coupled to themagnetic particles. The antibody is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available.

[0056] To practice the method, the AC133 antibody is added to a cellsample. The amount of AC133 Ab necessary to bind a particular cellsubset is empirically determined by performing a test separation andanalysis. The cells and AC133 antibody are incubated for a period oftime sufficient for complexes to form, usually at least about 5 min,more usually at least about 10 min, and usually not more than one hr,more usually not more than about 30 min.

[0057] The cells may additionally be incubated with antibodies orbinding molecules specific for cell surface markers known to be presentor absent on progenitor or stem cells.

[0058] The labeled cells are separated in accordance with the specificantibody preparation. Fluorochrome labeled antibodies are useful forFACS separation, magnetic particles for immunomagnetic selection,particularly high gradient magnetic selection (HGMS), etc. Exemplarymagnetic separation devices are described in WO 90/07380,PCT/US96/00953, and EP 438,520. The AC133 Cell Isolation Kit (MiltenyiBiotec Inc., Auburn Calif.) can be used for the positive selection ofAC133⁺ cells. The kit provides a tool for single step isolation ofAC133⁺ cells. The AC133 Cell Isolation Kit contains FcR Blocking Reagentand MACS colloidal MicroBeads conjugated to the monoclonal mouseanti-human AC133 antibody.

[0059] The purified cell population may be collected in any appropriatemedium. Various media are commercially available and may be used,including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic SaltSolution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI,Iscove's modified Dulbecco's medium (IMDM), phosphate buffered saline(PBS) with 5 mM EDTA, etc., frequently supplemented with fetal calfserum (FCS), bovine serum albumin (BSA), human serum albumin (HSA), etc.

[0060] Populations highly enriched for human progenitor or stem cellsare achieved in this manner. The desired cells will be 30% or more ofthe cell composition, preferably 50% or more of the cell population,more preferably 90% or more of the cell population, and most preferably95% or more (substantially pure) of the cell population.

[0061] Use of purified stem cell/progenitor cells. The AC133⁺ stemcells/progenitor cells are useful in a variety of ways. The AC133⁺ cellscan be used to reconstitute a host whose cells have been lost throughdisease or injury. Genetic diseases associated with cells may be treatedby genetic modification of autologous or allogeneic stem cells tocorrect a genetic defect or treat to protect against disease.Alternatively, normal allogeneic progenitor cells may be transplanted.Diseases other than those associated with cells may also be treated,where the disease is related to the lack of a particular secretedproduct such as hormone, enzyme, growth factor, or the like. CNSdisorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include Alzheimer's Disease, MultipleSclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, andParkinson's Disease, have been linked to the degeneration of neuralcells in particular locations of the CNS, leading to the inability ofthese cells or the brain region to carry out their intended function. Byproviding for maturation, proliferation and differentiation into one ormore selected lineages through specific different growth factors theprogenitor cells may be used as a source of committed cells.Neurospheres can also be used to produce a variety of blood cell types,including myeloid and lymphoid cells, as well as early hematopoieticcells (see, Bjornson et al., 283 SCIENCE 534 (1999), incorporated hereinby reference).

[0062] The AC133⁺ cells may also be used in the isolation and evaluationof factors associated with the differentiation and maturation of cells.Thus, the cells may be used in assays to determine the activity ofmedia, such as conditioned media, evaluate fluids for growth factoractivity, involvement with dedication of lineages, or the like.

[0063] The AC133⁺ cells may be frozen at liquid nitrogen temperaturesand stored for long periods of time, being thawed and capable of beingreused. The cells will usually be stored in 5% DMSO and 95% fetal calfserum. Once thawed, the cells may be expanded by use of growth factorsor stromal cells associated with stem cell proliferation anddifferentiation.

[0064] The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. These examplesshould in no way be construed as limiting the scope of the invention, asdefined by the appended claims.

EXAMPLE 1 AC133 Magnetic Cell Sorting (MACS) Positive Selected FetalBrain Cells Contain Neurosphere Initiating Cell (NS-IC) Activity

[0065] AC133⁺ cells are prepared by the following method: Human fetalbrain (FBR 10-20 gestational week [“g.w.”]) were obtained from AdvancedBioscience Resources, INC (Oakland, Calif.) after obtaining informedconsent. Human fetal brain tissues were cut into 1-3 mm cubed piecesusing scalpels, transferred into 50 mL centrifuge tubes and wash oncewith 0.02% EDTA/PBS solution. Tissues were dissociated enzymatically inthe presence of collagenase and hyaluronidase at 37° C. for 1 hr. Debrisand aggregates were removed by filtering cell suspensions through 70micron filter cup.

[0066] AC133⁺ human fetal brain cells were separated by usingparamagnetic antibody microbeads, AC133/1 Cell isolation Kit (Cat. #508-01, Miltenyi Biotec, Auburn, Calif.). MACS separations wereperformed based on instruction accompanied with the kit. In arepresentative flow cytometric MACS separation from a typical AC133⁺isolation, about 44% of the cells were AC133⁺ CD45⁻, while about 2% wereCD34⁺ (These CD34⁺ cells were endothelial cells complexed to thepurified NS-IC).

[0067] The AC133⁺ selected cells which resulted from the methoddescribed above (using brain 18 g.w.) were still heterogeneous. Thecells tended to form a complex with endothelial cells. Endothelial cellswere identified as CD34⁺ or CD105⁺. AC133 MACS separation also enrichesCD34⁺ endothelial cells which are associated with AC133⁺ cells. (Afterpassaging, the NS-IC separate from the complexed endothelial cells andpurified NS-IC can be obtained.)

[0068] AC133⁺ MACS separated cells were cultured in the presence ofmedia containing EGF, FGF-2, and LIF, as described above. In general,cells from early gestational age fetal brain (5-12 g.w.) were enrichedfor NS-IC and no enrichment was required to initiate neurospherecultures. On the other hand, cells from older fetal brain samples (16-20g.w.) contained far less NS-IC activity and required enrichment forinitiating neurosphere cultures. In other words, AC133⁺ is useful forthe enrichment of NS-IC from older gestational age humans brain tissue.AC133⁺ MACS separated cells from fetal brain (18 g.w.) were enriched forNS-IC activity, while whole human fetal brain cells (18 g.w.) withoutAC133⁺ MACS separation failed to initiate neurosphere cultures.

[0069] Neurosphere cells established from AC133 MACS cells expressnestin, as tested after ˜7 days in culture and detected by rabbitanti-human nestin polyconal antibodies. For example, among theneurosphere cells available from CytoTherapeutics (Providence, R.I.),FBR 1069 (18 g.w.) and FBR 1070 (20 g.w.) expressed nestin. When inducedto differentiate, the AC133⁺ MACS-derived neurosphere cells coulddifferentiate into neurons and astrocytes, as detected by β-tubulinstaining for neurons and GFAP staining for astrocytes. In thisparticular differentiation assay, neurosphere cells were cultured onto alaminin-coated surface in the presence of 1% FBS and without EGF, FGF-2and LIF.

[0070] Other differentiation assay can be used to induce differentiationof NS-IC to neurons, astrocytes and oligodendrocytes.

EXAMPLE 2 AC133 is a Critical Cell Surface Marker Expressed on CellsFrom Long-Term Neurosphere Culture

[0071] A long-term neurosphere cell culture, 8.5 FBR, was obtained fromCytoTherapeutics Inc. (Providence, R.I.). The 8.5 FBR neurosphere cellsexpress AC133 relatively uniformly. These 8.5 FBR cells are also Thy-1⁺,CD166⁺, and HLA-DR⁺. When Ex Vivo 15 was used as basal media, higherpercentage of neurosphere cultures initiated from primary brain tissuefrom 18 g.w. It is therefore possible to evaluate AC133⁺ fraction ofcells in developing neurosphere cultures. The proportion of AC133⁺ cellsincreased as neurosphere developed. Once neurosphere cells were wellestablished, virtually all cells forming neurospheres expressed AC133.

EXAMPLE 3 Neurosphere Initiating Cells Can be Separated Using MonoclonalAntibody, AC133: Flow Cytometry Cell Sorting (FACS) Approach

[0072] The purpose of this EXAMPLE is to test whether AC133⁺ cells werethe only cells in the brain that have pluripotent NSC activity. mAbagainst human CD45 was used to exclude blood cell contamination in fetaltissue. In some cases, mAb against human CD34 was also used to excludeendothelial cells and endothelial-neural progenitor complexes. The fetalbrain cells were thus defined as CD45⁻ CD34⁻. To measure neural stemcells and primitive progenitor activities, a NS-IC assay was establishedto determine frequency of NS-IC in a given population. When NS-IC arerare and express AC133 antigen uniformly, NS-IC can be enriched byAC133⁺ selection, and correspondingly depleted in other fractions.

[0073] Source of monoclonal antibodies: AC133 antigen was defined by twomAbs AC133/1 and AC133/2, both conjugated with phycoerythrin, which areavailable through Miltenyi Biotec (Auburn, Calif.). Anti human CD45-FITCand Glycophrin A-FITC were obtained from CALTAG (Burlingame, Calif.) andCoulter (Miami, Fla.) respectively. Anti-humanallophycocyanin-conjugated CD34 was obtained from BDIS (San Jose,Calif.).

[0074] Cell preparation: FBR were dissociated by collagnease andhyaluronidase, and still contained endothelial-progenitor complex, whichprevented the isolation of a candidate of NSC in single cell suspension(endothelial cells are CD45⁺). To dissociate this endothelial cell-NS-ICcomplex, FBR cells processed as described above, were further treatedwith trypsin for 10-15 min. The AC133 antigen, CD45 antigen and CD34antigen were resistant for trypsin treatment, while Glycophrin A wassensitive.

[0075] After trypsin digestion, cells were washed and stained with mAbsagainst CD45, Glycophrin A, AC133 and CD34. No immunomagnetic beadselections were used. The cells were incubated for 20-60 min on ice.After the final wash, the cells were resuspended in HBSS solutioncontaining 1 μg/mL propidium idodine (PI). The labeled cells wereanalyzed and sorted with a dual-laser FACS (Becton Dickinson, San Jose).Dead cells were excluded from analysis by their PI stainingcharacteristics. After sorting purity of sorted cell populations werechecked by FACS reanalysis. A representative FACS profiles of beforesorting and post-sorting of AC133⁺ CD45⁻ cells (NS-IC, ˜5% of thestarting cells) and AC133⁻ CD45⁻ cells (˜87% of the starting cells) wasperformed.

[0076] NS-IC activity is highly enriched in the AC133⁺ but not theAC133⁻ subset: FBR cells (typically 16-20 g.w.) were typically sortedfor CD45⁻ CD34⁻ AC133⁻ and CD45⁻ CD34⁻ AC133⁺ fractions. No significantNS-IC activity resided in CD45⁺ or CD45⁻CD34⁺ populations in FBR.

[0077] The sorted cells were cultured in the neurosphere media describedabove. Typically, Ex Vivo 15 or combination of Ex Vivo 15, D-MEM, F-12media were used as a basal medial. To maximize neurosphere development,the sorted cells were typically cultured in the presence of LIF, FGF-2,EGF and neural survival factor, NSF (Cat. CC-4323, Clonetics, San Diego,Calif.).

[0078] A single cell suspension was obtained after cell sorting. After4-5 days in vitro culture, the AC133⁺ cells started to proliferate andsmall neurospheres were observed 8-10 days post culture initiations. Thecells could initiate neurospheres when cultured in the presence of LIF,FGF-2, EGF without NSF. Neurosphere cultures were initiated from fourout of four FBR tissues (18-20 g.w.) sorted for AC133⁺ CD45⁻ or AC133⁺CD45⁻ CD34⁻.

[0079] In contrast, when AC133⁻ CD45⁻ FBR cells were placed in culturein the presence of LIF, FGF-2, and EGF, very few neurosphere formationswere seen, and failed to passage to a new flask. When additional NSF wasadded in the growth media, some neurosphere initiation was observed.Thus, AC133⁻ CD45⁻ FBR cells were depleted in a significant amount ofNS-IC.

EXAMPLE 4 AC133⁺ Cell Separation to Enrich for NS-IC Cells

[0080] AC133⁺ cell separation can effectively be used to enrich forNS-IC cells from tissue. Furthermore AC133⁺ cell separation can furtherenrich for NS-IC cells from established preparations. In one test,AC133⁺ cell sorting of dissociated neurospheres (CytoTherapeutics,Providence, R.I.) provides a greatly enriched NS-IC culture and showsincreased neurosphere establishment. Using that culture, the cell doserequired to initiate a neurosphere in each well (i.e., 100% positive)can be reduced from 3,000-10,000 cells to about 30 cells (see, TABLE 1,below). TABLE 1 Cell % % Tissue ID Cell Dose Score #Well PositiveNegative FBR 1104 Post trypsin 1,000 6 24 25.0% 75.0% Ex Vivo 15 3,00020 24 83.3% 16.7% LIF/EGF/ 10,000 24 24 100.0% 0.0% FGF-2 30,000 12 12100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1104 Post trypsin 1,000 23 2495.8% 4.2% Ex/Vivo 15 3,000 24 24 100.0% 0.0% LJF/EGF/ 10,000 24 24100.0% 0.0% FGF-2/NSF 30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0%FBR 1104 AC133 neg. 1,000 0 24 0.0% 100.0% selected cells Ex Vivo 153,000 1 24 4.2% 95.8% LIF/EGF/ 10,000 28 48 58.3% 41.7% FGF-2/NSF AC133⁺10 2 24 8.3% 91.7% selected cells Ex Vivo 15 100 24 24 100.0% 0.0%LIF/EGF/ 300 24 24 100.0% 0.0% FGF-2/NSF 1,000 24 24 100.0% 0.0% FBR1101 Post trypsin 1,000 9 24 37.5% 62.5% Ex Vivo 15 3,000 16 24 66.7%33.3% LIF/EGF/ 10,000 23 24 95.8% 4.2% FGF-2 30,000 12 12 100.0% 0.0%100,000 12 12 100.0% 0.0% FBR 1101 Post trypsin 1,000 16 24 66.7% 33.3%Ex Vivo 15 3,000 21 24 87.5% 12.5% LIF/EGF/ 10,000 24 24 100.0% 0.0%FGF-2/NSF 30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1101AC133⁺ 1 9 96 9.4% 90.6% selected cells Ex Vivo 15 10 42 60 70.0% 30.0%LIF/EGF/ 30 23 24 95.8% 4.2% FGF-2/NSF 100 11 12 91.7% 8.3%

[0081] As shown in TABLE 1, the non-enriched fresh brain tissue (“FBR”)used here (g.w. 20) may contain NS-IC in such numbers that it requires acell dose of between 3,000 and 10,000 cells to initiate neurospheres inevery well. By using the method of the invention, enriched populationscan be obtained, such that a cell dose of 1,000 cells or less isrequired, and more preferably an enriched population such that a celldose of less than 100 cells is required. As shown in TABLE 1, enrichmenthere has been achieved so that a cell dose of only about 30 cells isrequired per well to establish a neurosphere culture in each well. TABLE1 also shows that when populations are depleted in AC133⁺ cells (FBR1104 AC133 neg. selected cells), establishment of neurosphere culturesfrom those populations is markedly reduced.

[0082] Quantitative NS-IC assay: To assay for the presence of NS-IC,cell populations suspected of containing the multipotent NS-IC aresubjected to clonal development. Cells are grown in proliferation mediumto form neurospheres, then induced to differentiate to form neurons,astrocytes, and oligodendrocytes. The presence of neurons, astrocytes,and oligodendrocytes can be shown by immunostaining. For example,neurons stain for the presence of β-tubulin; astrocytes stain for thepresence of GFAP; and oligodendrocytes stain for the presence of O4.

[0083] The quantitative NS-IC assay can be performed on unpurifiedtissue cells, on AC133⁺ sorted cells, and on clonal neurosphere celllines.

EXAMPLE 5 Cell Culture Media For Growth and Passage of NS-IC

[0084] Weiss et al., U.S. Pat. No. 5,750,376 and Weiss et al., U.S. Pat.No. 5,851,832 disclose “culture medium containing one or morepredetermined growth factors effective for inducing multipotent neuralstem cell proliferation” and “differentiation-inducing conditions”However, different basal media can be used, including, but not limitedto:

[0085] D-MEM/F12 (Gibco BRL, Gaithersburg, Md.);

[0086] Ex Vivo 15 (Bio Whittaker, Walkersville, Md.);

[0087] Neural progenitor basal media, (Clonetics. San Diego, Calif.); or

[0088] combination of the basal media listed above.

[0089] A typical media formulation to culture human neurosphere cells isprovided in TABLE 2 Serum-Free N2/EGF Supplemented Culture Medium ForNeurospheres Quantity Reagents 87 ml DMEM/F12 (Gibco lot. 1012915; Cat.No. 11330-032) 1 ml N-2 Supplement (Gibco lot 1017018; Cat. No.17502-014) 1 ml 0.2 mg/ml heparin (Sigma lot 28H0320; Cat. No. H-3149) 1ml 0.2M Glutamine (JCR lot 7N2320; Cat. No. 59202-77p) 10 ml 3% Glucose(Sigma, lot 37H0841; Cat. No. G-7021) 20 μl 100 μg/ml EGF (R&D lotCE107091; Cat. No. 236-EG) 100 μl 20 μg/ml FGF-2 (Gibco lot KCQ411; Cat.No. 13256-029) 100 μl 10 μg/ml LIF (R&D lot OX038021; Cat. No. 250-L)

[0090] EGF is added to 100 ml base medium for human neurospheres afterfiltering the medium. EGF is relatively stable in the medium. FGF-2 andLIF are added when medium is ready to use. The final concentrations ofthe supplement reagents are: 5 μg/ml Insulin 100 μg/ml Human transferrin6.3 ng/ml Progesterone 16.1 μg/ml Putrascine 5.2 ng/ml Selenite 20 ng/mlEGF 20 ng/ml FGF-2 10 ng/ml LIF 2 μg/ml heparin 2 mM L-glumtamine 6mg/ml Glucose

[0091] The optimization of media formulation permits a higher percentageof neurospheres initiated from primary brain tissue to be established.We prefer Ex Vivo 15 media. The optimization of media formulation alsopermits a more consistent growth of neurospheres. To maximizeneurosphere development, the NS-IC are typically cultured in thepresence of LIF, bFGF, EGF and neural survival factor, NSF (Cat.CC-4323, Clonetics, San Diego, Calif.). In one test, both trypsinizedFBR 1101 neural cells and trypsinized FBR 1104 neural cells(CytoTherapeutics, Providence, R.I.), show increased growth whencultured in Ex Vivo 15 medium with LIF, bFGF, EGF, and NSF.

EXAMPLE 6 Direct Isolation of Human Neural Stem Cells From Fetal BrainBy Cell Sorting

[0092] A large source of highly defined engraftable human cells capableof extensive neuronal regeneration could be an effective therapeuticproduct for the treatment of neurodegenerative disorders. To definereproducible methods for the enrichment of human neural stem cells(NSCs), we have developed and used monoclonal antibodies (mAbs) directedtoward surface markers on human neural cells to identify and purify NSCsby fluorescence activated cell sorting (FACS). Based on FACS andimmunohistochemical analyses, two mAbs, 5F3 and 5E12 were identified.They defined small subsets of fetal brain cells and displayed specificreactivity to cells in the floor plate and ependymal layer of the spinalcord (12 g.w), sites known to contain CNS stem cells. These mAbs, stainless than 5% of FBR cells, and greater than 95% of cells from long-termneurosphere cultures were positive.

[0093] As an example, two cell populations 5F3+CD34−CD45−(5F3+) and5F3−CD34−CD45−(SF3−) were sorted and tested for their ability toinitiate neurosphere cultures. The 5F3+ subset was highly enriched forneurosphere-initiating cell activity; they proliferated to form smallneurosphere by 8-10 days in culture. In contrast, the sorted 5F3− cellsremained as a single cell suspension, failed to initiate neurospheres,and eventually died. The expanded 5F3+ sorted neurosphere cells werepositive for nestin expression, and differentiated into neurons and gliafollowing exposure to differentiation conditions. Using the NOD SCIDmouse, in vivo studies show that at 8 weeks post transplantation the5F3+ neurosphere cells can engraft and migrate. These studies show thatwe have identified and enriched human NSCs based on cell surface markersand flow cytometry and demonstrated their activity using in vitro andvivo assays.

[0094] In further experiments, we examined brain and spinal cord tissuesover various gestational ages. The earlier (5-12 wk gestation)gestational ages have a higher frequency of neurosphere initiating cell(NS-IC) than later gestational ages (16-20 wk gestation). See, e.g.,FIG. 5. Direct culturing of cells derived from these tissues leads toneurosphere initiation.

[0095] Our data (shown in the Table below) demonstrate that cellpopulation of neural cells enriched for 5F3⁺ cells are enriched forNS-IC activity, as much as 23 fold. Population % in brain NS-IC RangePost processed 100 1/819 1/304-1435 brain cells (n = 8) 5F3 − sorted (n= 6) 95 1/5434 1/4224-7772 5F3 + sorted (n = 6) 4.6 1/36 1/10-74

[0096] Further, as FIG. 2 shows, neurospheres can be derived fromsingle-cell sorted 5F3⁺ cells. We have also demonstrated thatself-renewal of neurosphere cells derived from 5F3⁺ sorted cells can beachieved by re-initiation of neurospheres from single cells (data notshown). Conversely, our data indicates that cell populations depleted of5F3⁺ cells are also depleted for NS-IC activity.

EXAMPLE 7 Isolation of NS-IC By Different Markers

[0097] As a second example, we sorted cell populations using a novelmonoclonal antibody, 5E12, described herein. The 5E12⁺ subset wasenriched for neurosphere-initiating cell activity, as shown in the Tablebelow. See also FIG. 3. Our data suggests that the antigen to the 5E12antibody is coexpressed with the AC133antigen on 5F3⁺ cells.

[0098] We also evaluated the 8G1 monoclonal antibody as a subselectorfor neural stem cells, as shown in the Table below. Cells that were 5F3⁺and 8G1^(-/lo) displayed more stem cell-like properties, while cellsthat were 5F3⁺ and 8G1^(med/hi) displayed more progenitor cell-likeproperties. ENRICHMENT OF NS-IC BY 5F3, 5E12 AND 8G1 ANTIBODIESPopulation % in brain NS-IC Range Brain cells 100 1/819 1/304-1435control (n = 8) 5F3 − sorted (n = 6) 95 1/5434 1/4224-7772 5F3 + (n = 6)4.6 1/36 1/10-74 5E12 − (n = 2) 97 1/1335 1/1259,1411 5E12 + (n = 3) 2.51/286 1/79-392 5F3 + 8G1^(−/lo) (n = 3) 1.1 1/23 1/15-34 5F3 +8G1^(mid/hi) (n = 3) 1.7 1/63 1/38-105

EXAMPLE 9 In Vivo Studies NS-IC

[0099] We transplanted 5F3+ sorted NS-ICs (obtained as described above)into the lateral ventricles of neonatal immunodeficient mice, usingconventional techniques. Engraftment and migration of human neurospherecells were detected between 4-8 weeks after injection using a humanspecific Thy-1 antibody (see FIG. 6). As shown in FIG. 7, staining withhuman β-tubulin (a neuronal marker) and human nuclear antigen (forlocalization of human cells) revealed migration of the human neurospherecells through the rostral migratory stream (RMS). Further, as shown inFIG. 8, localization using human nuclear antigen demonstrated that humanneurosphere cells had migrated through the RMS to the olfactory bulb.

[0100] The foregoing description has been presented only for thepurposes of illustration and is not intended to limit the invention tothe precise form disclosed, but by the claims appended hereto.

We claim:
 1. A method for producing a population highly enriched forhuman central nervous system stem cells (CNS-SC) which can initiateneurospheres (NS-IC), comprising: (a) contacting a population containingneural or neural-derived cells with a reagent that recognizes adeterminant on a cell surface marker recognized by monoclonal antibodyAC133 or by monoclonal antibody 5E12; and (b) selecting for cells inwhich there is contact between the reagent and the determinant on thesurface of the cells of step (a), to produce a population highlyenriched for CNS-SC.
 2. The method of claim 1, wherein the reagent is anantibody that recognizes a determinant on the cell surface markerrecognized by monoclonal antibody AC133.
 3. The method of claim 2,wherein the reagent is an AC133 monoclonal or polyclonal antibody. 4.The method of claim 2, wherein the reagent is a ligand or small moleculethat binds to the surface marker recognized by an AC133 antibody or a5E12 antibody.
 5. The method of claim 1, wherein the reagent is anantibody that recognizes a determinant on the cell surface markerrecognized by a 5E12 monoclonal antibody.
 6. The method of claim 5,wherein the reagent is a 5E12 monoclonal or polyclonal antibody.
 7. Themethod of claim 1, wherein the reagent is fluorochrome conjugated. 8.The method of claim 1, wherein the reagent is conjugated to magneticparticles.
 9. The method of claim 1, wherein the selecting is by flowcytometry.
 10. The method of claim 1, wherein the selecting is byfluorescence activated cell sorting or high gradient magnetic selection.11. The method of claim 1, wherein the selecting is by a physicalpositive selection device.
 12. The method of claim 1, wherein thepopulation containing neural or neural-derived cells is obtained fromany tissue which gives rise to neural tissue.
 13. The method of claim 1,wherein the population containing neural or neural-derived cells isdissociated from neural tissue.
 14. The method of claim 1, wherein thepopulation containing neural or neural-derived cells is derived from afetal brain, adult brain, fetal spinal cord or adult spinal cord. 15.The method of claim 1, wherein the population containing neural orneural-derived cells is obtained from a neural cell culture.
 16. Themethod of claim 1, wherein the population containing neural orneural-derived cells is obtained from a neurosphere culture or anadherent monolayer.
 17. The method of claim 1, further comprising: (c)contacting the population containing neural or neural-derived cells witha reagent that binds to CD45 antigens; and (d) selecting for cells inwhich there is contact between the cells and the reagent that recognizesa determinant on a cell surface marker recognized by monoclonal antibodyAC133 or by monoclonal antibody 5E12 and selecting for reduced contactbetween the cells and the reagent that binds to CD45 antigens, such thatthose cells that are AC133⁺ CD45⁻ or 5E12⁺ CD45⁻ or AC133⁺ 5E12⁺ CD45⁻are selected.
 18. The method of claim 1, further comprising: (c)contacting the population containing neural or neural-derived cells witha reagent that binds to CD45 antigen; (c) contacting the populationcontaining neural or neural-derived cells with a reagent that binds toCD34 antigen; and (b) selecting for cells in which there is contactbetween the cells and the reagent that recognizes a determinant on acell surface marker recognized by monoclonal antibody AC133 or bymonoclonal antibody 5E12 and a reduced contact between the cells and thereagent that binds to CD45 antigen, and between the cells and thereagent that binds to CD34 antigen, such that those cells that areAC133⁺ CD45⁻ CD34⁻ or 5E12⁺ CD45⁻ CD34⁻ or AC133⁺ 5E12⁺ CD45⁻ CD34⁻ areselected.
 19. The method of claim 1, further comprising: (c) contactingthe population containing neural or neural-derived cells with a reagentthat recognizes a determinant on a cell surface marker recognized bymonoclonal antibody 8G1; and (b) selecting for cells in which there iscontact between the cells and the reagent that recognizes a determinanton a cell surface marker recognized by monoclonal antibody AC133 or bymonoclonal antibody 5E12 and a reduced contact between the cells and thereagent that recognizes a determinant on a cell surface markerrecognized by monoclonal antibody 8G1, such that those cells that areAC133⁺ 8G1^(-/lo) or 5E12⁺ 8G1^(-/lo) or AC133⁺ 5E12⁺ 8G1^(-/lo) areselected.
 20. A method for enriching a population of neural cells forthe population's neurosphere initiating stem cell (NS-IC) fraction,comprising: (a) combining a population comprising neural cells orneural-derived cells containing a fraction of NS-ICs with a reagent thatrecognizes a determinant on a cell surface marker recognized bymonoclonal antibody AC133 or by monoclonal antibody 5E12; and (b)selecting for AC133⁺ and/or 5E12⁺ cells, wherein the selected AC133⁺ or5E12⁺ cells are enriched in the fraction of NS-IC as compared with thepopulation of neural cells.
 21. A method for identifying a neurosphereinitiating stem cell (NS-IC), comprising: (a) contacting a populationcontaining neural or neural-derived cells with a reagent that recognizesa determinant on a cell surface marker recognized by monoclonal antibodyAC133 or by monoclonal antibody 5E12; and (b) detecting the contactbetween a cell of the population containing neural or neural-derivedcells and the reagent that recognizes a determinant on a cell surfacemarker recognized by monoclonal antibody AC133 or by monoclonal antibody5E12, wherein an identification of a cell as being AC133⁺ or 5E12⁺identifies the cell as an NS-IC.
 22. A method for isolating aneurosphere initiating stem cell (NS-IC), comprising: (a) combining apopulation comprising neural cells or neural-derived cells containing afraction of NS-ICs with a reagent that recognizes a determinant on acell surface marker recognized by monoclonal antibody AC133 or bymonoclonal antibody 5E12; (b) selecting AC133⁺ or 5E12⁺ cells, whereinthe selected AC133⁺ or 5E12⁺ cells are enriched in the fraction ofNS-ICs as compared with the population of neural cells; (c) introducingat least one AC133⁺ or 5E12⁺ cell to a culture medium capable ofsupporting the growth of NS-IC; and (d) proliferating the AC133⁺ or5E12⁺ cell in the culture medium.
 23. A population of cells produced bythe method of claim
 22. 24. The method of claim 22, wherein the culturemedium capable of supporting the growth of NS-IC comprises a serum-freemedium containing one or more predetermined growth factors effective forinducing and/or permitting multipotent neural stem cell proliferation.25. The method of claim 24, wherein the culture medium capable ofsupporting the growth of NS-IC further comprises a growth factorselected from the group consisting of leukocyte inhibitory factor (LIF),epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2)and combinations thereof.
 26. The method of claim 25, wherein theculture medium capable of supporting the growth of NS-IC furthercomprises a neural survival factor (NSF).
 27. An in vitro cell culturecomposition comprising: (a) a population enriched in AC133⁺ CD45⁻ cellsor 5E12⁺ CD45⁻ cells; and (b) a medium capable of supporting the growththe cells.
 28. An in vitro cell culture composition comprising: (a) apopulation enriched in AC133⁺ CD45⁻ CD34⁻ cells or 5E12⁺ CD45⁻ CD34⁻cells; and (b) a medium capable of supporting the growth the cells. 29.An in vitro cell culture composition comprising: (a) a populationenriched in AC133⁺ 8G1^(-/lo) cells or 5E12⁺ 8G1^(-/lo) cells; and (b) amedium capable of supporting the growth the cells.
 30. An in vitro cellculture composition comprising: (a) a population enriched in AC133⁺8G1^(hi) cells or 5E12⁺ 8G1^(hi) cells; and (b) a medium capable ofsupporting the growth the cells.
 31. An in vitro cell culturecomposition comprising: (a) a population comprising at least 50% AC133⁺or 5E12⁺ neurosphere initiating cells (NS-IC) which stain positive fornestin and, in the presence of differentiation-inducing conditions,produce progeny cells that differentiate into neurons, astrocytes, andoligodendrocytes; and (b) a medium capable of supporting the growth ofNS-IC.
 32. The composition of claim 31, further comprising a solidsupport to which the NS-IC are attached.
 33. The composition of claim31, wherein the population of cells has at least 70% AC133⁺ or 5E12⁺cells.
 34. The composition of claim 31, wherein the population of cellshas at least 90% AC133⁺ or 5E12⁺ cells.
 35. The composition of claim 31,wherein the population of AC133⁺ or 5E12⁺ cells is a substantially purepopulation.
 36. The composition of claim 31, wherein the mediumcomprises a serum-free medium containing one or more predeterminedgrowth factors effective for inducing multipotent neural stem cellproliferation.
 37. The composition of claim 31, wherein the mediumfurther contains a growth factor selected from the group consisting ofleukocyte inhibitory factor (LIF), epidermal growth factor (EGF), basicfibroblast growth factor (FGF-2), and combinations thereof.
 38. Thecomposition of claim 31, wherein the medium further comprises a neuralsurvival factor.
 39. The composition of claim 31, wherein the NS-IC arehuman.
 40. A method for characterizing a neurosphere initiating stemcell (NS-IC), comprising: (a) introducing an isolated AC133⁺ or 5E12⁺cell to a culture medium capable of supporting the growth of NS-IC; (b)proliferating the AC133⁺ or 5E12⁺ cells in the culture medium; (c)culturing the progeny of the isolated AC133⁺ or 5E12⁺ cell underconditions in which the isolated AC133⁺ cell or 5E12⁺ celldifferentiates to neurons, astrocytes, and oligodendrocytes; and (d)detecting the presence of neurons, astrocytes, and oligodendrocytes,wherein the presence of neurons, astrocytes, and oligodendrocytescharacterizes the isolated AC133⁺ cell or 5E12⁺ cell as an NS-IC. 41.The method of claim 40, wherein the conditions in which the isolatedAC133⁺ or 5E12⁺ cell progeny differentiates to neurons, astrocytes, andoligodendrocytes comprises culturing the progeny of the isolated AC133⁺or 5E12⁺ cell on a laminin-coated surface in culture medium with fetalbovine serum (FBS) and without mitogenic growth factor.
 42. A method foridentifying the presence of a growth factor that affects the growth of aneurosphere initiating stem cell (NS-IC), the method comprising: (a)combining a composition suspected of containing at least one growthfactor that affects the growth of NS-IC with a composition comprisingNS-IC, wherein the NS-IC are characterized as AC133⁺ or 5E12+, nestin⁺,and capable of differentiation to neurons, astrocytes, andoligodendrocytes lineages; and (b) determining the growth of the NS-ICas a function of the presence of at least one of the growth factors inthe composition, wherein an altered NS-IC growth, as compared withgrowth of NS-IC that have not been contacted with the compositionsuspected of containing at least one growth factor that affects thegrowth of NS-IC, indicates the presence in the composition of a growthfactor that affects the growth of NS-IC.
 43. The method of claim 42,wherein the growth factor is a neural survival factor.